We have developed a simple and accurate biosensor based on a chemiluminescent (CL)-lateral flow immunoassay (LFIA) method integrated in a smartphone to quantitatively detect salivary cortisol. The ...biosensor is based on a direct competitive immunoassay using peroxidase–cortisol conjugate, detected by adding the chemiluminescent substrate luminol/enhancer/hydrogen peroxide. The smartphone camera is used as light detector, for image acquisition and data handling via a specific application. We 3D-printed simple accessories to adapt the smartphone. The system comprises a cartridge, which houses the LFIA strip, and a smartphone adaptor with a plano-convex lens and a cartridge-insertion slot. This provides a mini-darkbox and aligned optical interface between the camera and the LFIA membrane for acquiring CL signals. The method is simple and fast, with a detection limit of 0.3ng/mL. It provides quantitative analysis in the range of 0.3–60ng/mL, which is adequate for detecting salivary cortisol in the clinically accepted range. It could thus find application in the growing area of home-self-diagnostic device technology for clinical biomarker monitoring, overcoming the current difficulties in achieving sensitive and quantitative information with conventional systems taking the advantage of smartphone connectivity and the enhanced performance of the included camera.
•A biosensor based on chemiluminescent lateral flow immunoassay was developed.•Smartphone's camera was used as a detector for chemiluminescent signals.•Smartphone's accessories were realized for acquiring chemiluminescent signals.•The biosensor allowed to quantify cortisol in saliva sample.
In this paper, we report, for the first time, the use of a smartphone to image and quantify biochemiluminescence coupled biospecific enzymatic reactions to detect analytes in biological fluids. Using ...low-cost three-dimensional (3D) printing technology, we fabricated a smartphone accessory and a minicartridge for hosting biospecific reactions. As a proof-of-principle, we report two assays: a bioluminescence assay for total bile acids using 3α-hydroxyl steroid dehydrogenase coimmobilized with bacterial luciferase system and a chemiluminescence assay for total cholesterol using cholesterol esterase/cholesterol oxidase coupled with the luminol–H2O2–horseradish peroxidase system. These assays can be performed within 3 min in a very straightforward manner and provided adequate analytical performance for the analysis of total cholesterol in serum (limit of detection (LOD) = 20 mg/dL) and total bile acid in serum and oral fluid (LOD = 0.5 μmol/L) with a reasonable accuracy and precision. Smartphone-based biochemiluminescence detection could be thus applied to a variety of clinical chemistry assays.
Increasingly, smartphones are used as portable personal computers, revolutionizing communication styles and entire lifestyles. Using 3D-printing technology we have made a disposable minicartridge ...that can be easily prototyped to turn any kind of smartphone or tablet into a portable luminometer to detect chemiluminescence derived from enzyme-coupled reactions. As proof-of-principle, lactate oxidase was coupled with horseradish peroxidase for lactate determination in oral fluid and sweat. Lactate can be quantified in less than five minutes with detection limits of 0.5 mmol L
−1
(corresponding to 4.5 mg dL
−1
) and 0.1 mmol L
−1
(corresponding to 0.9 mg dL
−1
) in oral fluid and sweat, respectively. A smartphone-based device shows adequate analytical performance to offer a cost-effective alternative for non-invasive lactate measurement. It could be used to evaluate lactate variation in relation to the anaerobic threshold in endurance sport and for monitoring lactic acidosis in critical-care patients.
Smartphone-based chemiluminescence biosensor for lactate detection.
•Smartphone-interfaced biosensors represent a very promising research field.•Bioluminescence whole-cell biosensors can be exploited as reliable analytical tools for toxicity assays.•A 3D printed ...smartphone-based bioluminescence whole-cell toxicity biosensor is reported.
In this work, we report the design, fabrication, and preliminary assessment of analytical performance of a smartphone-based bioluminescence (BL) whole-cell toxicity biosensor. Genetically engineered human embryonic kidney cells, constitutively expressing a powerful green-emitting luciferase mutant, were used as “sentinel cells” and integrated into 3D printed ready-to-use cartridges, also containing assay reagents. Customizable, low-cost smartphone adaptors were created using a desktop 3D printer to provide a mini-darkbox and an aligned optical interface between the smartphone camera and the cell cartridge for BL signals acquisition. The developed standalone compact device, which also includes disposable droppers for sample and reagents addition, allows the user to perform the toxicity assay within 30min following the procedure provided by a custom-developed application running on Android (Tox-App). As proof-of-concept we analyzed real samples including ubiquitous products used in everyday life. The results showed good correlation with those obtained with laboratory instrumentation and commercially available toxicity assays, thus supporting potential applications of the proposed device for portable real-life needs.
In recent years, there has been an increasing demand for predictive and sensitive in vitro tools for drug discovery. Split complementation assays have the potential to enlarge the arsenal of in vitro ...tools for compound screening, with most of them relying on well-established reporter gene assays. In particular, ligand-induced complementation of split luciferases is emerging as a suitable approach for monitoring protein-protein interactions. We hereby report an intracellular nanosensor for the screening of compounds with androgenic activity based on a split NanoLuc reporter. We also confirm the suitability of using 3D spheroids of Human Embryonic Kidney (HEK-293) cells for upgrading the 2D cell-based assay. A limit of detection of 4 pM and a half maximal effective concentration (EC
) of 1.7 ± 0.3 nM were obtained for testosterone with HEK293 spheroids. This genetically encoded nanosensor also represents a new tool for real time imaging of the activation state of the androgen receptor, thus being suitable for analysing molecules with androgenic activity, including new drugs or endocrine disrupting molecules.
Whole-cell biosensors present many advantages, including being able to monitor the toxicity and bioavailability of chemicals; cells grown in traditional 2D cultures, however, do not reproduce the ...complexity of in vivo physiology. In the last years, 3D cell-culture models have garnered great attention due to their capability to better mimic in vivo cellular responses to external stimuli, providing excellent model living organisms. In order to obtain a predictive, sensitive, and robust yet low-cost 3D cell biosensor, we developed a smartphone-based bioluminescent 3D cell biosensor platform for effect-based analysis. We exploited the Nuclear Factor-kappa B (NF-kB) signal transduction pathway, which is induced by several types of stressors and is involved in the regulation of cell-cycle/growth, inflammation, apoptosis, and immunity. The smartphone-based biosensor relies on immobilized HEK293 spheroids genetically engineered with powerful red- and green-emitting luciferases utilized as inflammation and viability reporters. It provides a limit of detection for Tumor Necrosis Factor (TNFα) of 0.15 ± 0.05 ng/mL and could be a useful tool to initially screen environmental samples or other compounds on-site, especially for additional more accurate chemical analyses.
•We developed a smartphone-based bioluminescent biosensor for inflammatory activity.•The biosensor employs a 3D printed cartridge with immobilized spheroids of human cells.•Red- and green-emitting luciferases are used as inflammation and viability reporters.•Potential applications of the biosensor include environmental and food analysis.
The availability of smartphones with high-performance digital image sensors and processing power has completely reshaped the landscape of point-of-need analysis. Thanks to the high maturity level of ...reporter gene technology and the availability of several bioluminescent proteins with improved features, we were able to develop a bioluminescence smartphone-based biosensing platform exploiting the highly sensitive NanoLuc luciferase as reporter. A 3D-printed smartphone-integrated cell biosensor based on genetically engineered Hek293T cells was developed. Quantitative assessment of (anti)-inflammatory activity and toxicity of liquid samples was performed with a simple and rapid add-and-measure procedure. White grape pomace extracts, known to contain several bioactive compounds, were analyzed, confirming the suitability of the smartphone biosensing platform for analysis of untreated complex biological matrices. Such approach could meet the needs of small medium enterprises lacking fully equipped laboratories for first-level safety tests and rapid screening of new bioactive products.
Graphical abstract
Smartphone-based bioluminescence cell biosensor
Firefly luciferases, which emit visible light in a highly specific ATP-dependent process, have been adapted for a variety of applications, including gene reporter assays, whole-cell biosensor ...measurements, and in vivo imaging. We previously reported the approximately 2-fold enhanced activity and 1.4-fold greater bioluminescence quantum yield properties of a chimeric enzyme that contains the N-domain of Photinus pyralis luciferase joined to the C-domain of Luciola italica luciferase. Subsequently, we identified 5 amino acid changes based on L. italica that are the main determinants of the improved bioluminescence properties. Further engineering to enhance thermal and pH stability produced a novel luciferase called PLG2. We present here a systematic comparison of the spectral and physical properties of the new protein with P. pyralis luciferase and demonstrate the potential of PLG2 for use in assays based on the detection of femtomole levels of ATP. In addition, we compared the performance of a mammalian codon-optimized version of the cDNA for PLG2 with the luc2 gene in HEK293T cells. Using an optimized low-cost assay system, PLG2 activity can be monitored in mammalian cell lysates and living cells with 4.4-fold and approximately 3.0-fold greater sensitivity, respectively. PLG2 could be an improved alternative to Promega’s luc2 for reporter and imaging applications.
Beetle luciferases have been adapted for live cell imaging where bioluminescence is dependent on the cellular availability of ATP, O2, and added luciferin. Previous Photinus pyralis red-emitting ...variants with high Km values for ATP have performed disappointingly in live cells despite having much higher relative specific activities than enzymes like Click Beetle Red (CBR). We engineered a luciferase variant PLR3 having a Km value for ATP similar to CBR and ∼2.6-fold higher specific activity. The red-emitting PLR3 was ∼2.5-fold brighter than CBR in living HEK293T and HeLa cells, an improvement consistent with the importance of the Km value in low ATP environments.
The availability of new bioluminescent proteins, obtained by cDNA cloning and mutagenesis of wild-type genes, expanded the applicability of these reporters from the perspective of using more proteins ...emitting at different wavelengths in the same cell-based assay. By spectrally resolving the light emitted by different reporter proteins, it is in fact possible to simultaneously monitor multiple targets. A new luciferase isolated from Luciola italica has been recently cloned, and thermostable red- and green-emitting mutants were obtained by random and site-directed mutagenesis. Different combinations of luciferases were used in vitro as purified proteins and expressed in bacterial and mammalian cells to test their suitability for multicolor assays. A mammalian triple-color reporter model system was then developed using a green-emitting wild-type Photinus pyralis luciferase, a red thermostable mutant of L. italica luciferase, and a secreted Gaussia princeps luciferase (GLuc) to monitor the two main pathways of bile acid biosynthesis. The two firefly luciferases were used to monitor cholesterol 7-α hydroxylase and sterol 27-hydroxylase, while secreted constitutively expressed GLuc was used as an internal vitality control. By treating the cells with chenodeoxycholic acid, it was possible to obtain dose-dependent inhibitions of the two specific signals together with a constant production of GLuc, which allowed for a dynamic evaluation of the metabolic activity of the cells. This is the first triple-color mammalian reporter assay that combines secreted and nonsecreted luciferases requiring different substrates, thus avoiding reciprocal interference between different BL signals. This approach is suitable for high content analysis of gene transcription in living cells to shorten the time for screening assays, increasing throughput and cost-effectiveness.
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